The activation of gonadotropin-releasing hormone (GnRH) neurons at puberty is not well understood but recent evidence suggests a role for the kisspeptin-KISS1R system as a key regulator of reproductive development and function. While it is clear that kisspeptin and KISS1R function as essential regulators of the neuroendocrine cascade, many questions remain about the role of this novel ligand-receptor system in the regulation of GnRH neuronal function at the time of puberty as well as during subsequent reproductive function. The overarching hypothesis underlying this project is that intracellular signal transduction by kisspeptin is influenced by desensitization, internalization, and recycling/degradation of the KISS1R, thereby playing critical roles in controlling the timing of puberty and maintaining cyclical reproductive function. An additional hypothesis for this project is that a better understanding of the mechanisms by which mutations affect the kisspeptin-KISS1R system will have clinical implications by providing insights into the pathophysiology of the associated phenotypes, laying the groundwork for the development of new diagnostic tools as well as for genetic counseling of patients and their family members, and identifying novel therapeutic targets. We propose the following specific aims: (1) To characterize KISS1R-mediated intracellular signaling, trafficking, turnover, desensitization, and resensitization; (2) To identify extracellular co-factors that modulate KISS1R signaling and to study their mechanisms of action in the regulation of kisspeptin-mediated KISS1R activation; and (3) To generate and characterize a KISS1R (R386P) knock-in mouse as a model to better understand the mechanisms by which this mutation is associated with central precocious puberty (CPP). The successful completion of the proposed studies will advance our understanding of the kisspeptin/KISS1R system in the regulation of GnRH neuronal function at the time of puberty as well as during subsequent reproductive function and discover potential therapeutic targets for patients with reproductive disorders. Furthermore, completion of these studies will provide training in the generation of genetically manipulated mouse models and will enrich the Candidate's research experience in experimental design, aiding in the development of a successful independent translational investigator. PUBLIC HEALTH RELEVANCE: The mechanisms underlying the activation of the reproductive cascade at puberty are not well understood. Recent evidence suggests a role for the kisspeptin-KISS1R system as a key regulator of reproductive development and function. The successful completion of the proposed research will advance our understanding of this novel ligand-receptor system in the regulation of puberty onset, one of the greatest mysteries of human biology, and identify therapeutic targets for infertility, which affects one in seven couples.